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Interleukin-12-based local gene therapy of cancer constitutes an active area of clinical research using plasmids, mRNAs and viral vectors. To improve antitumor effects, we have experimentally tested the combination of mRNA constructs encoding interleukin-12 (IL-12) and interleukin-18 (IL-18). Moreover, we have used a form of IL-18 (DR-18) which has preserved bioactivity but does not bind to the IL-18BP decoy receptor. Both cytokines dramatically synergize to induce IFNγ release from mouse splenocytes, and, if systemically co-transferred to the liver, they mediate lethal toxicity. However, if given intratumorally to B16OVA tumor-bearing mice, the combination attains efficacy against the directly treated tumor and moderate tumor-delaying activity on distant uninjected lesions. Co-treatment was conducive to the presence of more activated CD8+ T cells in the treated and non-injected tumors. In keeping with these findings, the efficacy of treatment was contingent on the integrity of CD8+ T cells and cDC1 dendritic cells in the treated mice. Furthermore, efficacy of IL-12 plus DR-18 local mRNA co-injection against distant concomitant tumors could be enhanced upon combination with anti-PD-1 mAb systemic treatment, thus defining a feasible synergistic immunotherapy strategy.

BackgroundModified vaccinia virus Ankara (MVA) are genetically engineered non-replicating viral vectors. Intratumoral administration of MVA induces a cyclic GMP-AMP synthase-mediated type I interferon (IFN) response and the production of high levels of the transgenes engineered into the viral genome such as tumor antigens to construct cancer vaccines. Although type I IFNs are essential for establishing CD8-mediated antitumor responses, this cytokine family may also give rise to immunosuppressive mechanisms.MethodsIn vitro assays were performed to evaluate the activity of simvastatin and atorvastatin on type I IFN signaling and on antigen presentation. Surface levels of IFN α/β receptor 1, endocytosis of bovine serum albumin-fluorescein 5 (6)-isothiocyanate, signal transducer and activator of transcription (STAT) phosphorylation, and real-time PCR of IFN-stimulated genes were assessed in the murine fibroblast cell line L929. In vivo experiments were performed to characterize the effect of simvastatin on the MVA-induced innate immune response and on the antitumor effect of MVA-based antitumor vaccines in B16 melanoma expressing ovalbumin (OVA) and Lewis lung carcinoma (LLC)-OVA tumor models. RNAseq analysis, depleting monoclonal antibodies, and flow cytometry were used to evaluate the MVA-mediated immune response.ResultsIn this work, we identified commonly prescribed statins as potent IFNα pharmacological inhibitors due to their ability to reduce surface expression levels of IFN-α/β receptor 1 and to reduce clathrin-mediated endocytosis. Simvastatin and atorvastatin efficiently abrogated for 8 hours the transcriptomic response to IFNα and enhanced the number of dendritic cells presenting an OVA-derived peptide bound to major histocompatibility complex (MHC) class I. In vivo, intraperitoneal or intramuscular administration of simvastatin reduced the inflammatory response mediated by peritumoral administration of MVA and enhanced the antitumor activity of MVA encoding tumor-associated antigens. The synergistic antitumor effects critically depend on CD8+ cells, whereas they were markedly improved by depletion of CD4+ lymphocytes, T regulatory cells, or NK cells. Either MVA-OVA alone or combined with simvastatin augmented B cells, CD4+ lymphocytes, CD8+ lymphocytes, and tumor-specific CD8+ in the tumor-draining lymph nodes. However, only the treatment combination increased the numbers of these lymphocyte populations in the tumor microenvironment and in the spleen.ConclusionIn conclusion, blockade of IFNα functions by simvastatin markedly enhances lymphocyte infiltration and the antitumor activity of MVA, prompting a feasible drug repurposing.